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ORIGINAL PAPER
Physiological acclimation of two psammophytes to repeated soildrought and rewatering
Yayong Luo • Xueyong Zhao • Ruilian Zhou •
Xiaoan Zuo • Jinghui Zhang • Yanqing Li
Received: 21 November 2009 / Revised: 11 April 2010 / Accepted: 7 May 2010
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010
Abstract To understand physiological acclimation of
psammophyte to repeated soil drought and rewatering, two
psammophytes (Setaria viridis and Digitaria ciliaris) were
subjected to three cycles of soil drought and rewatering.
The response process of leaf relative water content (RWC),
membrane permeability, lipid peroxidation, gas exchange
characteristics, antioxidant enzymes, soluble protein, and
free proline was examined. Leaf RWC, the net photosyn-
thesis rate, stomatal conductance, and water use efficiency
decreased, while membrane permeability, lipid peroxida-
tion, intercellular CO2 concentration, soluble protein, and
free proline increased during three soil drought periods for
both psammophytes. These physiological characteristics
were recovered to the control levels following rewatering
for 4 days. However, activities of SOD, CAT, and POD
were induced continuously under soil drought conditions,
and remained higher than those in the control throughout
the whole experiment period, which agrees with our
hypothesis that drought hardening activates defensive
systems of both psammophytes continuously. Decreasing
level of leaf RWC and increasing levels of leaf membrane
permeability and lipid peroxidation were suppressed with
increasing the number of drought cycles, suggesting that
drought hardening alleviates damages of both psammo-
phytes and improves their drought tolerance and acclima-
tion to soil drought conditions in the future. Additionally,
the photosynthesis decreased more slowly in the sub-
sequent drought cycles than in the first cycle, allowing both
psammophytes to maximize assimilation in response to
repeated soil drought conditions. Thus, both psammophytes
acclimatize themselves to repeated soil drought.
Keywords Photosynthesis � Antioxidant enzymes �Proline � Recovery � Hardening
Introduction
Water belongs to the most important resource for plant life,
and is associated with various physiological processes of
plants. The water availability in most arid and semi-arid
ecosystems usually does not meet plant demand (Chen
et al. 2005). Furthermore, the water availability is spatially
and temporally heterogeneouson (Huxman et al. 2004).
As a result, plants are repeatedly exposed to drought
during their life cycles due to continuous changes in cli-
matic factors in these regions (Miyashita et al. 2005). It is
important to understand the mechanisms that trigger
off physiological responses to drought and rehydration
conditions.
Water is the crucial limiting factor for plant recruitment,
photosynthesis, growth, and net ecosystem productivity
(Weltzin and McPherson 2000; Xu et al. 2007) due to its
severely restricted supply in arid ecosystems. Hence, an
arid ecosystem tends to show the effects of precipitation
Communicated by R. Aroca.
Y. Luo � X. Zhao � X. Zuo � J. Zhang � Y. Li
Cold and Arid Regions Environmental and Engineering
Research Institute, Chinese Academy of Sciences,
No. 320 Donggang Road, Lanzhou 730000, Gansu, China
Y. Luo (&)
Graduate University of Chinese Academy of Sciences,
Beijing 100049, China
e-mail: [email protected]; [email protected]
R. Zhou
Department of Biological Science and Biotechnology,
Ludong University, Yantai 264025, China
123
DOI 10.1007/s11738-010-0519-5
Acta Physiol Plant (2011) 33:79–91
/ Published online: 201016 June
variation rapidly (Xu et al. 2007). Responses of crops and
trees exposed to soil drought and rewatering conditions
have been well documented (Dichio et al. 2006; Liang and
Zhang 1999; Ortuno et al. 2005; Zhang et al. 2004). Little,
however, is known about the strategies of the psammo-
phyte during frequent water shortage and rewatering
cycles. As a matter of fact, most of these studies applied
only one drought and rewatering cycles to their experi-
ments (Liang and Zhang 1999). It is difficult to understand
the true responses to repeated drought and rewatering
processes when only one drought cycle is applied. There-
fore, how do plants respond to repeated soil drought and
rewatering conditions, which has been paid little attention,
deserves further studies. Moreover, vegetative growth of
stressed plants can recover after rewatering (Liang and
Zhang 1999; Ortuno et al. 2005), suggesting a reversibility
of physiological changes generated by water deficiency.
Soil drought can create oxidative stress in photosynthetic
organisms, which causes oxidative injury to plant cells by
increased production of reactive oxygen species (ROS)
(Asada 1999; Gong et al. 2006). To defend against oxi-
dative stress, plants often induce or enhance activities of
various antioxidant enzymes such as superoxide dismutase
(SOD), catalase (CAT), and peroxidase (POD) (Asada
2006; Gong et al. 2006). Additionally, plant accumulates
low molecular compounds, such as soluble protein, free
proline to alleviate both cellular hyperosmolarity and ion
disequilibrium (Handa et al. 1986; Jiang and Huang 2002;
Parida et al. 2007; Echevarria-Zomeno et al. 2009) in
drought periods.
The Horqin Sandy Land is one of the most seriously
desertification-threatened areas in China (Andren et al.
1994). Plants in this area often survive during longer soil
drought period and recover upon precipitations, but little
information exists on the physiological mechanism
involved. Setaria viridis and Digitaria ciliaris are the
dominant annuals in sandy land (Li et al. 2005); as a
result, they are good candidates to employ for vegetation
restoration initiatives. A study of their responses to
repeated water shortages and rewatering conditions may
contribute to understanding physiological acclimation of
psammophyte. The responses of leaf RWC, membrane
permeability, lipid peroxidation, gas exchange charac-
teristics, ROS-scavenging enzymes systems, soluble
protein and free proline of these two species to repeated
soil drought and rewatering processes were therefore
examined.
Yordanov et al. (2003) reported that repair processes
lead also to hardening of plants by establishing a new
physiological standard, which is an optimum stage of
physiology under the changed environmental conditions.
Defensive systems can be activated continuously or
induced through exposure to oxidative stress (Buchanan
et al. 2000). We hypothesized that: Psammophytes defen-
sive systems can be activated continuously after drought
hardening, which may alleviate damages of psammophytes
and improve their drought tolerance and acclimation to soil
drought conditions in the future.
Materials and methods
Experimental design
The study was conducted at the south-western (42�550N,
120�440E; approximately 360 m ASL) Horqin Sandy
Land region representing the most desertification-threa-
tened area in North China (Andren et al. 1994). This area
has a temperate, semi-arid, and continental monsoonal
climate, receiving 360 mm annual mean rainfall, with
75% of it occurring between June and September.
Annual mean latent evaporation is 1,935 mm (Li et al.
2005). A number of psammophytes were dominant,
including S. viridis, D. ciliaris, Aristida adscensionis,
Salsola collina, Agriophyllum squrrosum, Cleistogenes
squarrosa, Chloris virgata, Caragana microphylla L.,
Lespedeza davurica, Artemisia halodendron and Arte-
misia frigida.
The plants, S. viridis and D. cilliaris, were selected
from sandy dune with uniform size (height of 20 cm) and
transplanted into plastic pots (diameter 22 cm, height
19 cm) with original soil (88.5% sand, 7.5% silt, and
4.0% clay) on 2 July 2008. Water content at saturation
and field capacity of the sandy soil was 21.03 and
13.07%, respectively. Thirty plants were nurtured to
recover from transplanted injury under natural conditions
with well irrigation until the onset of the experiments for
each species. Excess water was allowed to drain through
holes in bottoms of the pots. Experimental treatments
were put into practice on 13 August when leaf had been
completely developed and matured. Thirty plants were
subjected to two different treatments for each species,
well watered (control) and drought and rewatering
(drought stressed), arranged in a completely randomized
design (Galle et al. 2007). Control plants were then
continuously watered daily to field capacity during the
experimental period. Drought-stressed treatments were
carried out in three time periods for drought (from August
14 to 19, from August 23 to 29, and from September 2 to
8), alternating with rewatering (from August 19 to 22 and
from August 29 to September 1). All pots were placed
into a mobile rain shelter, which was drawn back to avoid
rainfall, and undrawn to ensure the natural climate con-
ditions after rain. Water-stressed plants were kept without
water until net photosynthesis approached zero during the
late morning (Galle et al. 2007).
123
Acta Physiol Plant (2011) 33:79–9180
Throughout the experiment, leaf relative water content
(RWC), relative electrolyte leakage (REL), leaf gas
exchange, anti-oxidative systems, and osmosis were
determined on natural expanded leaves at least five ran-
domly selected plants per treatment. Physiological mea-
surements were conducted on the sunny and windless days
(i.e., the first, third, and sixth days after watering excluding
days that were overcast or rainy) for three drought periods,
respectively. The three drought periods were interrupted by
two rewatering periods for 4 days. Water-treated scheme
and sampling days through experiment were shown in
Fig. 1.
Soil water status and climatic data
Changes in pot weight of drought-stressed and control
plants were monitored (Galle et al. 2007) to reflect soil
water status. Photosynthetic active radiation (PAR), air
temperature, and relative humidity of air were recorded
with a portable photosynthesis system (LI-6400, LI-COR
Inc., Lincoln, NE, USA) during gas exchange measure-
ments through experiment.
Sampling and gas exchange
On each sampling day, 5 g leaves collected at 10:00 AM
from at least five randomly selected plants per treatment
were mixed together and cut into segments of 2–3 cm.
The segments of leaves were divided into two parts
for each species: one used for testing leaf RWC and
REL; one stored with liquid N for lipid peroxidation,
antioxidant enzymes, soluble protein, and free proline
analysis.
The net photosynthetic rate (Pn), transpiration rate (Tr),
stomatal conductance (gs), and intercellular CO2 concen-
tration (Ci) were measured during 9:30–11:00 AM using
a portable photosynthesis system for both species. The
measurements were conducted on mature and expanded
leaves of five different plants per treatment, under
uniform conditions (30�C, 358–380 lmol (CO2) mol-1 and
1,500 lmol m-2 s-1 of PAR (provided by a built-in red
LED light source)) (Chen et al. 2005). The ratio of Pn to Tr
was calculated to determine instantaneous water use effi-
ciency (WUE).
Leaf relative water content (RWC) Fresh leaves (0.3 g)
were weighed quickly for determining leaf fresh weight
(FW). Then leaves were rehydrated by immersing the
petiole into distilled water in a petri dish capped with nylon
mesh. Full rehydration was achieved in 3 h, then blotted up
and weighed for leaf turgid weight (TW) determination.
Subsequently, dry weight (DW) was determined after
oven-drying leaf samples at 70�C for 24 h (Izanloo et al.
2008); five replicates per treatment were taken. Leaf RWC
was calculated according to the following equation:
RWC %ð Þ ¼ FW� DWð Þ � 100= TW� DWð Þ ð1Þ
Leaf membrane permeability It was quantified by
determining REL (Zhu et al. 2002). 0.3 g of fresh leaves
were placed into test tubes contained 20 cm3 of deionized
water; five replicates per treatment were taken. The initial
electrical conductivity was determined by measuring the
electrical conductivity (Cole-Parmer 19820, Cole-Parmer
Inc., Vernon Hills, IL, USA) of the water after 10 min of
applying a vacuum air pump (limit vacuum 26 kPa) and
3 h in a shaking incubator at room temperature. Then
the segment was boiled for 5 min, and allowed to cool
at room temperature prior to measuring the maximum
electrical conductivity. The REL was expressed as
the ratio of initial and maximum electrical conductivity
(Zhu et al. 2002).
Enzyme extraction Leaf samples were grounded in
liquid nitrogen and mixed with chilled extraction buffer
(50 mM phosphate, 1% (w/v) polyvinylpolypyrrolidone).
The extract was filtered through gauze and centrifuged at
15,000g at 4�C for 10 min. The resulting supernatant was
stored at 4�C for assaying malondialdehyde (MDA) con-
tent, activities of SOD, CAT, and POD, soluble protein,
and free proline content. The extract experiments were
repeated two times independently (with three replicates of
each extract sample); thus, each data point was the mean of
six replicates (n = 6) (Demiral and Turkan 2005).
Lipid peroxidation It was estimated by measuring the
malondialdehyde (MDA) content using the thiobarbituric
acid method described by Zhao et al. (1994). 5 cm3 of
Fig. 1 Changes in photosynthetic active radiation (PAR) (columns)
and relative humidity of air (line with triangles) for each sampling
day through experiment. Drought-stressed plants were rewatered on
6–9 and 16–19 days (hatched areas). Means and SE of five replicates
are shown
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Acta Physiol Plant (2011) 33:79–91 81
0.5% thiobarbituric acid and 3 cm3 of extraction superna-
tant were mixed together and heated for 10 min at 100�C
and then cooled. The homogenate was centrifuged at
10,000g for 10 min, and the supernatant was measured at
532, 450, and 600 nm. Leaf MDA content (nmol ml-1)
was determined as 6.45(A532 - A600) - 0.56A450, where
A532, A600, and A450 denotes absorbance in 532, 600, and
450 nm, respectively.
Antioxidant enzymes The assay for total SOD (EC1.15.1.1)
activity was based on the method according to Wang et al.
(2009). One unit of SOD activity is defined as the amount
of enzyme required to inhibit the reduction of nitroblue
tetrazolium (NBT) by 50%. The determination of CAT
activity was done using iodine–starch method according to
Zhao et al. (1994). One unit of CAT activity is defined as
the amount of enzyme required to reduce 10-6 mol H2O2
per minute. POD activity was measured by following the
change in absorption at 470 nm due to guaiacol oxidation
(Reuveni et al. 1992). One unit of POD activity
is expressed as the increases of absorbance of 0.01 per
minute. The enzyme activity for three enzymes was
expressed as units (U) per gram of fresh weight (FW)
(Tan et al. 2008).
Soluble protein concentrations They were determined
according to the principle of protein dye binding described
by Bradford (1976), using bovine serum albumin as a
standard. The soluble protein concentrations in samples
were expressed as milligram per gram of FW.
Free proline content It was determined based on the
method of Bates et al. (1973). After addition of acid nin-
hydrin and glacial acetic acid, resulting mixture was heated
at 100�C for 40 min in water bath. Reaction was then
stopped by using ice bath. The mixture was extracted with
toluene, and the absorbance of fraction with toluene aspired
from liquid phase was read at 520 nm. Free proline content
was determined using calibration curve and expressed as
lg proline per gram of FW.
The above colorimetric assay was measured by using
UV-1601 UV–Visible Spectrophotometer (Shimadzu
Corporation, Japan).
Statistic analysis
Students t test analyses on independent samples of leaf gas
exchange parameters (n = 5), REL (n = 5), MDA content
(n = 6), antioxidant enzymes activities (n = 6), soluble
protein (n = 6), and free proline content (n = 6) were
performed, respectively, testing for significant differences
between water stressed and control for two species on each
day measurements were taken (Galle et al. 2007) using the
SPSS 13.0 software. Pearson correlation was used to reflect
relations among leaf RWC and the physiological charac-
teristics for the two drought-stressed plants, respectively.
Results
Environmental conditions and water status
The climatic conditions during the experimental periods
were typical of summer in Horqin Sandy Land. Photo-
synthetic active radiation, the relative humidity, and air
temperature on sampling days through experiment ranged
between 1,103 and 1,604 lmol m-2 s-1, 26.6 and 37.2%
(Fig. 1), and 29.3 and 30.4�C, respectively (Fig. 2).
Ambient CO2 concentration ranged between 358 and
380 lmol (CO2) mol-1 (data not shown) throughout the
whole experimental period.
During drought periods, the loss of soil water was
reflected in the progressive decline of pot weight (Fig. 3a, b).
The minimal pot weight was reached after 6 days during
the first drought period and 7 days during the second and
third drought periods. It remained unchanged during re-
watering periods for 4 days. Control plants showed little
changes in pot weight throughout the whole experimental
period. After rewatering, soil water status was restored
immediately (Fig. 3a, b). According to the depletion of soil
water, Leaf RWC in drought-stressed plants decreased with
ongoing soil drought stress and dropped to 29.38, 42.49,
and 41.10% for S. viridi (Fig. 3c) and 31.99, 37.47, and
44.18% for D. ciliaris (Fig. 3d) on the last days of three
drought periods, respectively. Leaf RWC was restored to
the control level after rewatering following rewatering for
4 days (Fig. 3c, d). Control plants showed slight changes in
the leaf RWC throughout the experimental period, ranging
Fig. 2 Changes in air temperature for each sampling day through
experiment. Drought-stressed plants were rewatered on 6–9 and 16–
19 days (hatched areas). Means and SE of five replicates are shown
123
Acta Physiol Plant (2011) 33:79–9182
between 70.40 and 90.29% for S. viridis (Fig. 3c), and
between 75.37 and 89.53% for D. ciliaris (Fig. 3d). Thus,
decreasing level of leaf RWC diminished with increasing
the number of drought cycles.
Leaf relative electrolyte leakage and malondialdehyde
content
Control plants showed slight changes in the leaf REL and
MDA content throughout the experimental period, ranging
between 5.95 and 10.11%, and between 2.92 and
4.24 nmol g-1FW for S. viridis (Fig. 4a, c), and ranging
between 6.88 and 13.61%, and between 2.4 and
2.9 nmol g-1FW, respectively, for D. ciliaris (Fig. 4b, d).
Leaf REL and MDA content increased with ongoing soil
drought, but they declined to or near the control levels
following rewatering for 4 days. Leaf REL increased by
5.15 times for S. viridi and 5.40 times for D. ciliaris in the
first drought cycle and increased by 1.63 and 1.57 times for
S. viridi, and 1.30 and 0.64 times for D. ciliaris in the
sequent cycles. Leaf MDA content increased by 65.42,
27.10, and 17.84% for S. viridi and increased by 53.37,
24.76, and 28.23% in three drought periods, respectively,
for D. ciliaris. Therefore, increasing levels of leaf REL and
MDA content were suppressed through repetitions of
soil drought. Additionally, leaf REL was often lower in
Fig. 3 Changes in the water
status of soil (pot weight) and
leaf RWC during repeated soil
drought and rewatering for
S. viridis (a, c) and D. ciliaris(b, d), respectively. Opencircles and filled circles denote
the control and drought-stressed
plants, respectively. Drought-
stressed plants were rewatered
on 6–9 and 16–19 days
(hatched areas). Means and SE
of pot weight (n = 15 pots) and
Leaf RWC (n = 5) are shown
Fig. 4 Changes in relative
electrolyte leakage (REL) and
malondialdehyde (MDA) during
repeated soil drought and
rewatering in S. viridis (a, c)
and D. ciliaris (b, d),
respectively. Open circles and
filled circles denote the control
and drought-stressed plants,
respectively. Drought-stressed
plants were rewatered on 6–9
and 16–19 days (hatchedareas). Means and SE of REL
(n = 5) and MDA content
(n = 6) are shown. Significant
difference between well-
watered and stressed plants at
each date: *P B 0.05;
**P B 0.01; ***P B 0.001
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Acta Physiol Plant (2011) 33:79–91 83
S. viridis than in D. ciliaris under drought conditions,
whereas MDA content was higher in S. viridis (Fig. 4).
Gas exchange characteristics
The values of leaf Pn, gs, Ci, and WUE of control plants for
both species ranged predominantly between 10 and
20 lmol CO2 m-2 s-1, 0.1 and 0.15 mol H2O m-2 s-1,
103 and 143 lmol mol-1, and 4.0 and 5.5 mmol
CO2 mol-1 H2O for both species, respectively (Fig. 5).
Soil drought led to a progressive suppression of leaf Pn, gs,
and WUE, while they increased to the control levels
following rewatering for 4 days (Fig. 5). Leaf Ci
increased with the soil water shortage, especially on the
last days of drought periods, but it decreased to the
control levels following rewatering for 4 days. Addi-
tionally, leaf Pn, gs, and WUE declined more rapidly in
the first drought cycle than in subsequent cycles. For
instance, Pn was 19.07, 1.56, and 0.96 for three soil
moisture conditions in the first drought cycle for S. viri-
dis, but it was 13.87, 6.92, and 1.30 in the second cycle.
Changes in WUE (Fig. 5g, h) had a tendency similar to
that seen in Pn for both species. Leaf WUE decreased
during the drought period where reductions were pri-
marily recovered after irrigation was restored. Leaf WUE,
however, was always higher for control plants than
drought-stressed plants. Averaged WUE was 4.70, 2.78,
and 1.65 for S. viridis and 4.83, 3.30, and 2.07 for
D. ciliaris in all three soil moisture conditions for
drought-stressed plants while it was 4.79, 5.30, and 4.77
for S. viridis and 4.85, 5.27, and 4.97 for D. ciliaris,
respectively, for the control plants.
Activity of ROS-scavenging enzymes
The activities of SOD, CAT, and POD (Fig. 6) ranged
predominantly from 300 to 450 U g-1 FW, 400–
500 U g-1 FW, and 330–450 U g-1 FW for control
Fig. 5 Changes in Pn, gs, Ci, and WUE during repeated soil drought
and rewatering in S. viridis (a, c, e, g) and D. ciliaris (b, d, f, h). Opencircles and filled circles denote the control and drought-stressed
plants, respectively. Drought-stressed plants were rewatered on 6–9
and 16–19 days (hatched areas). Means and SE of five plants are
shown. Significant difference between well-watered and stressed
plants at each date: *P B 0.05; **P B 0.01; ***P B 0.001
123
Acta Physiol Plant (2011) 33:79–9184
S. viridi, respectively, and ranged predominantly from 160
to 270 U g-1 FW, from 150 to 270 U g-1 FW, and from
40 to 60 U g-1 FW for the control D. ciliaris, respectively.
SOD, CAT, and POD activities of drought-stressed plants
increased significantly in three drought periods, which
were higher in drought-stressed plants than in control
through experiment for both species (Fig. 6). Additionally,
levels of SOD, CAT, and POD activities were higher in
S. viridis than in D. ciliaris throughout whole experiment,
respectively (Fig. 6).
Soluble protein and free proline content
Control plants showed slight changes in soluble protein and
free proline content throughout the experimental period,
between 10.68 and 14.08 mg g-1 FW, and 43.18 and
127.15 lg g-1 FW, respectively, for S. viridis (Fig. 7a, c);
and between 8.09 and 9.77 mg g-1 FW, and 18.25 and
31.70 lg g-1 FW, respectively, for D. ciliaris (Fig. 7b, d).
Soluble protein and free proline content increased during
soil drought periods, while they decreased to the control
levels following rewatering for 4 days. Their increases
were slight on the intermediate days of drought periods
except for soluble protein in D. ciliaris, but remarkable on
the last days of drought periods. Soluble protein was 16.22,
16.01, and 14.63 for S. viridis and 13.91, 14.77, and 12.48
for D. ciliari on the last days of three drought periods,
respectively. Free proline increased by a factor of 32.72 for
S. viridis and by a factor of 56.40 for D. ciliari in the first
drought cycle and increased by a factor of 8.94 and 3.86 for
S. viridis and by a factor of 12.91 and 7.83 for D. ciliari in
subsequent cycles. Thus, increasing levels of free proline
diminished with increasing the number of drought cycles
(Fig. 7c, d). Additionally, the levels of soluble protein and
free proline were often higher in S. viridis than D. ciliari in
control plants, respectively (Fig. 7).
Discussion and conclusion
Drought damage, recovery, and photosynthetic
adjustment
Leaf RWC, REL, and MDA content indicate the extent of
dehydration, membrane permeability, and lipid peroxida-
tion, respectively. They are used to assess cellular damage
(Demiral and Turkan 2005; Bai et al. 2006; Gong et al.
2006). Leaf RWC is a valid index of the water equilibrium
in plants (Gong et al. 2006), and is used to assess the
severity of drought (Flexas and Medrano 2002). A con-
tinuous and slight decrease in leaf RWC levels was
observed in control plants during the experimental period
(Fig. 3c, d), likely owing to dry environment such as low
relative humidity (Fig. 1) and vapor pressure deficit when
water loss through the cuticle is substantial (Mott
and Parkhurst 1991; Bunce 2006), and leaf aging. The
Fig. 6 Changes in the activities of SOD, CAT, and POD in the leaves
of S. viridis (a, c, e) and D. ciliaris (b, d, f) during repeated soil
drought and rewatering. Open circles and filled circles denote the
control and drought-stressed plants, respectively. Drought-stressed
plants were rewatered on 6–9 and 16–19 days (hatched areas). Means
and SE of six replicates are shown. Significant difference between
well-watered and stressed plants at each date: *P B 0.05;
**P B 0.01; ***P B 0.001
123
Acta Physiol Plant (2011) 33:79–91 85
decreases coincide with changes in membrane permeabil-
ity, lipid peroxidation, gas exchange parameters, and
antioxidant enzymes in control plants (Figs. 3, 4, 5, 6)
(Dhindsa et al. 1981; Vos and Oyarzun 1987; Munne-
Bosch and Alegre 2004; Bunce 2006; Barron-Gafford et al.
2007). Leaf RWC decreased remarkably with ongoing soil
drought for both species for drought-stressed plants
(Fig. 3c, d). The content of MDA, produced during per-
oxidation of membrane lipids, is often used as an indicator
of oxidative damage (Demiral and Turkan 2005). Leaf
membrane permeability negatively correlated with leaf
RWC for both species, while MDA did not show signifi-
cant correlation with leaf RWC (Tables 1, 2). Leaf
membrane permeability and lipid peroxidation increased
with soil drought (Fig. 4), suggesting that cell membranes
were damaged (Gong et al. 2006; Wang and Li 2006). This
result was consistent with previous studies (Smirnoff and
Colombe 1988; Xu and Zhou 2006). In addition, leaf
membrane permeability was lower in S. viridis than in
D. ciliaris, whereas lipid peroxidation was higher in
S. viridis (Fig. 4c, d), indicating that S. viridis was sub-
jected to more oxidative damage than D. ciliaris. Fortu-
nately, the decreased levels of leaf RWC and the increased
level of leaf membrane permeability and lipid peroxidation
were suppressed with increasing the number of drought
cycles (Figs. 3, 4), suggesting that drought hardening
Fig. 7 Soluble protein and free
proline content during repeated
soil drought and rewatering in
S. viridis (a, c) and D. ciliaris(b, d). Open circles and filledcircles denote the control and
drought-stressed plants,
respectively. Drought-stressed
plants were rewatered on 6–9
and 16–19 days (hatchedareas). Means and SE of six
replicates are shown. Significant
difference between well-
watered and stressed plants at
each date: *P B 0.05;
**P B 0.01; ***P B 0.001
Table 1 Pearson correlation matrix of leaf RWC and physiological characteristics for drought-stressed S. viridi
Leaf RWC REL MDA Pn gs Ci WUE SOD CAT POD Protein
Leaf RWC 1
REL -0.97*** 1
MDA -0.24 0.08 1
Pn 0.76* -0.73* -0.22* 1
gs 0.81** -0.80* -0.02 0.96*** 1
Ci -0.73* 0.78* -0.10 -0.91*** -0.96*** 1
WUE 0.70* -0.75* 0.04 0.93*** 0.96*** -0.98*** 1
SOD -0.87** 0.75* 0.39 -0.84** -0.83** 0.72* -0.69* 1
CAT -0.52 0.61 -0.15 -0.77* -0.79* 0.87** -0.91*** 0.49 1
POD -0.83** 0.82** 0.19 -0.70* -0.75* 0.74* -0.60* 0.79* 0.65 1
Protein -0.57 0.68* -0.54 -0.62 -0.78* 0.83** -0.80* 0.39 0.68* 0.46 1
Proline -0.90*** 0.98*** 0.04 -0.61 -0.69* 0.70* -0.67* 0.60 0.55 0.73* 0.67*
* Correlation was significant at 0.05 level (two-tailed)
** Correlation was significant at 0.01 level (two-tailed)
*** Correlation was significant at 0.001 level (two-tailed)
123
Acta Physiol Plant (2011) 33:79–9186
alleviated extent of dehydration and enhanced cell mem-
brane stability under soil drought conditions for both spe-
cies (Bai et al. 2006; Gong et al. 2006; Villar-Salvador
et al. 2004; Yordanov et al. 2003), which agrees with the
hypothesis that drought hardening alleviate their damages
and improve drought tolerance and acclimation to soil
drought conditions in the future.
Leaf Pn in control plants (Fig. 5a, b) was comparable to
previous studies for oak trees in pots ranging between 10
and 20 lmol CO2 m-2 s-1 (Galle et al. 2007). Compared
to control plants, soil drought led to a rapid suppression of
Pn and gs (Fig. 5a–d) during soil drought periods, consis-
tent with previous studies (Souza et al. 2004; Galle and
Feller 2007). Leaf Pn and gs positively correlated with leaf
RWC for both species (Tables 1, 2). In addition, Pn and gs
decreased more slowly (Fig. 5a–d) with increasing the
number of drought cycles, indicating that photosynthetic
apparatus itself may enhance tolerant capacity to soil
drought in the future after drought hardening (Yordanov
et al. 2003). Therefore, psammophytes which experience
irregular water availability under field conditions have
evolved certain mechanisms to allow them to survive and
maximize assimilation in response to repeated soil drought
conditions.
Changes in gs indicate that closed stomata would open
following rewatering and then gradually closed again
during drought periods. As Pn decreased in parallel with gs
(Fig. 5a–d), stomatal limitation seemed to account for the
suppression of photosynthesis, especially on the interme-
diate days of drought periods. Stomatal closure protects
against further water loss and irreversible cell dehydration
under progressing soil drought conditions (Galle et al.
2007). On the other hand, Ci increased with the soil water
shortage, especially on the last days of drought periods
where gs was lower than 0.05 mol H2O m-2 s-1 (Fig. 5c–f),
and Ci negatively correlated with Pn and gs for both species
(Tables 1, 2). The overestimate Ci could result from het-
erogeneous (or ‘‘patch’’) stomatal closure and cuticular
conductance which are the two main problems invalidating
Ci calculation under drought, as decreasing mesophyll
conductance can cause the CO2 concentration in chloro-
plasts of stressed leaves to be considerably lower than Ci
(Flexas and Medrano 2002; Flexas et al. 2004b). Gunase-
kera and Berkowitz (1992) have reported that patchy CO2
assimilation pattern that occurs when bean plants are sub-
jected to a rapidly imposed stress could induce artifacts in
gas exchange studies, consistent with the present study. It
is concluded that decreases in photosynthesis may not only
indicate changes in the mesophyll capacity for photosyn-
thesis but may also be caused by heterogeneous stomatal
closure (Mott and Parkhurst 1991). Furthermore, the dra-
matic decreases in Pn and gs and sharp increases in Ci
(Fig. 5) suggest the predominance of non-stomatal limita-
tions to photosynthesis on the last days of drought periods
(Flexas and Medrano 2002; Flexas et al. 2004a), indicating
that photosynthetic apparatus of both psammophytes were
damaged, presumably due to decreases in photochemistry
and Rubisco activity (Flexas and Medrano 2002; Flexas
et al. 2006). The malfunction of photosynthetic apparatus
may reduce the efficiency of electron transport for photo-
synthetic reaction, which results in substantive accumula-
tion of ROS (Asada 1999; Garnczarska et al. 2004). ROS
can be generated by the direct transfer of the excitation
energy from chlorophyll to produce singlet oxygen or by
Table 2 Pearson correlation matrix of leaf RWC and physiological characteristics for drought-stressed D. ciliaris
Leaf RWC REL MDA Pn gs Ci WUE SOD CAT POD Protein
Leaf RWC 1
REL -0.86** 1
MDA 0.44 -0.33 1
Pn 0.91*** -0.74* 0.21 1
gs 0.97*** -0.84** 0.27 0.94*** 1
Ci -0.94*** 0.81** -0.27 -0.88** -0.97*** 1
WUE 0.93*** -0.73* 0.32 0.98*** 0.93*** -0.85** 1
SOD -0.82** 0.72* 0.031 -0.88** -0.84** 0.79* -0.86** 1
CAT -0.71* 0.49 0.06 -0.87** -0.76* 0.79* -0.81** 0.81** 1
POD -0.76* 0.67* 0.03 -0.77* -0.83** 0.80** -0.74* 0.89** 0.67* 1
Protein -0.88** 0.84** -0.35 -0.85** -0.90*** 0.87** -0.80** 0.80** 0.63 0.77* 1
Proline -0.91*** 0.95*** -0.40 -0.74* -0.90*** 0.91*** -0.73* 0.70* 0.52 0.72* 0.88**
* Correlation was significant at 0.05 level (two-tailed)
** Correlation was significant at 0.01 level (two-tailed)
*** Correlation was significant at 0.001 level (two-tailed)
123
Acta Physiol Plant (2011) 33:79–91 87
oxygen reduction in the Mehler reaction in chloroplast
(Stepien and Klobus 2005), and then promotes the mem-
brane lipid peroxidation in cell. Therefore, MDA content,
the product of lipid peroxidation, significantly increased
during soil drought periods (Fig. 4c, d).
Leaf RWC increased to the control level after watering
(Fig. 3c, d), which demonstrated that both psammophytes
respond rapidly to soil water irrigation, and restoration of
root water uptake was effective (Liang and Zhang 1999).
Leaf membrane permeability and lipid peroxidation
decreased to or near control levels after rewatering (Fig. 4),
indicating that injured cell membranes were alleviated and
recovered following rewatering (Xu and Zhou 2006).
Recovery was observed following rewatering where the
plants reached levels of Pn, gs, Ci, WUE, soluble protein,
and free proline similar to those in control plants, respec-
tively (Figs. 5, 7). Studies (Souza et al. 2004; Galle and
Feller 2007) have also reported that plants reached levels of
Pn and gs similar to those found in the control plants. In
contrast, Miyashita et al. (2005) reported that the fractional
recovery in Pn is higher than gs. Galle and Feller (2007)
reported that Pn recovered completely within 4 weeks for
stressed beech; meanwhile, gs remained permanently lower
in drought-stressed plants than it did in control plants.
Therefore, differences in recovery of Pn and gs may be
species-specific or stress-specific and requires further
investigations (Flexas et al. 2004a; Galle et al. 2007).
Potential higher WUE is due to stomatal closure (lower
gs) and decreases transpiration more than Pn (Hetherington
and Woodward 2003; Donovan et al. 2007). But higher
WUE comes at the cost of lower Pn and productivity
(Chaves et al. 2003; Donovan et al. 2007). Changes in
WUE had a tendency similar to leaf RWC (Figs. 3, 5).
WUE declined during soil drought periods (Fig. 5g, h), and
positively correlated with leaf RWC for both species
(Tables 1, 2). Moreover, intrinsic water use efficiency (Pn/
gs) decreased in three drought periods (data not shown);
thus, photosynthesis may be more restricted by the chlo-
roplast’s capacity to fix CO2 (metabolic limitations) than
by the increased diffusive resistance (Yordanov et al. 2000;
Flexas et al. 2004a), especially on the last days of drought
periods. Additionally, WUE in drought-stressed plants was
lower than those in control plants (Fig. 5g, h). As a result,
both psammophytes photosynthesized quickly and had high
WUE on the first days of drought periods, while suppres-
sion of photosynthesis and low WUE were observed during
later drought periods (Fig. 5a, b, g, h). The ‘‘drought
escape’’ strategy may therefore be adopted by both psam-
mophytes to cope with decreases of water availability
(Sherrard and Maherali 2006), allowing them to increase
their growth rate and accelerate development. Chaves et al.
(2002) reported that annuals often primarily rely on rapid
growth to escape drought stress as well as on fast
photosynthetic and C metabolism machinery responses in
semi-arid regions. This ‘‘fast growing’’ strategy allows
psammophytes to acclimatize themselves and compete
with other plants for available water in semi-arid sandy
land regions where a lack of precipitation occurs and water
is easily lost due to evaporation and percolation (Donovan
et al. 2007).
In general, suppression of photosynthesis is accompa-
nied by a downregulation of the photosynthetic activity
(i.e., PSII) and increased thermal dissipation of excess
excitation energy at midday, related to high temperature
and vapor pressure deficit (Epron et al. 1992). These effects
were reversible and vanished within minutes to hours after
relief of excessive light and presumably acting as a photo-
protective mechanism (Szabo et al. 2005). Water stress
amplified these effects: photosynthesis was strongly
decreased, showing important midday depression (Epron
et al. 1992), even induced irreversible damages within
green tissues because the formation of ROS may increase
under aggravated stress (Galle and Feller 2007). Plants may
activate and enhance their antioxidant enzymes such as
SOD, CAT, POD, and other compounds to protect them-
selves against irreversible damages (Asada 2006; Gong
et al. 2006).
Defensive mechanism
Lipid peroxidation significantly increased during three
drought periods (Fig. 4c, d), which stimulated to increase
activities of various antioxidant enzymes such as SOD,
CAT, and POD (Fig. 6) (Asada 2006; Gong et al. 2006).
SOD is considered to constitute the first line of defense
against ROS, which catalyzes superoxide radical (O2�-) to
O2 and H2O2 which are further scavenged by various
antioxidant enzymes (Demiral and Turkan 2005; Wang
et al. 2009; Zhang et al. 2004; Asada 2006), the most
important being CAT and POD (Gong et al. 2006). Reports
have shown that CAT is critical and indispensable for
maintaining the redox balance during oxidative stress
(Willekens et al. 1997). POD can act both as ROS scav-
enger and play multiple functions, because of its high
number of iso-forms (Passardi et al. 2005). The activities of
SOD, CAT, and POD remained higher than those in the
control after rewatering for 4 days (Fig. 6); as a result, they
were higher in drought-stressed plants than in control
throughout whole experimental period for both species
(Fig. 6). These results are consistent with the reports that
defensive systems can be activated continuously or induced
through exposure to soil drought stress (Buchanan et al.
2000; Mittler 2002; Zhang et al. 2004) to defend against
oxidants (Asada 1999; Gong et al. 2006), which agrees
with our hypothesis that drought hardening activates
psammophytes defensive systems continuously. Reparatory
123
Acta Physiol Plant (2011) 33:79–9188
processes lead to the hardening of plants by establishing a
new physiological standard, an optimum stage of physiol-
ogy under altered environmental conditions (Yordanov
et al. 2003). Additionally, the activities of SOD, CAT, and
POD are higher in S. viridis (Fig. 6a, c, e) than in D. cil-
iaris (Fig. 6b, d, f), probably because that S. viridis was
subjected to more oxidative damage than D. ciliaris.
Activities of SOD, CAT, and POD negatively correlated
with Pn (Tables 1, 2), suggesting that photosynthesis may
provide reducing power to help enzymatic detoxification
systems (Mittler 2002; Yordanov et al. 2003).
The alteration of protein synthesis or degradation is one
of the fundamental metabolic processes, which may
influence drought tolerance (Jiang and Huang 2002).
Accumulation of dehydrin protein was induced strongly by
severe drought stress, which could protect plants from
further dehydration during drought stress (Han and
Kermode 1996; Jiang and Huang 2002). In the present
study, soluble protein increased during three drought
periods (Fig. 7a, b), consistent with evidences of drought-
induced accumulation of proteins to water limitation (Bray
1993; Han and Kermode 1996; Jiang and Huang 2002).
However, drought-induced decrease in soluble protein
have also been reported in Bermuda grass (Barnett and
Naylor 1966), safflower (Carthamus mareoticus L.), and
cotton (Parida et al. 2007).
The function of proline as an osmoprotectant under
drought stress has been widely reported (Parida et al.
2008). In the present study, proline negatively correlated
with leaf RWC for both species (Tables 1, 2). Proline is
considered the principal solute, and its accumulation
showed a remarkable increase under drought conditions
(Fig. 7), which may allow both psammophytes to over-
come drought effect through osmotic adjustment and to
enhance their capacity of survival and tolerance under
drought conditions (Delauney and Verma 1993; Handa
et al. 1986). Furthermore, the accumulation of proline
contribute to enzyme protection, stabilization of biological
membranes, acclimation of photosynthetic apparatus,
storage of nitrogen and carbon for future use, scavenging
free radicals, storage energy to regulate redox potentials,
and recovery of stomata from the water shortage (Klein and
Itai 1989; Yordanov et al. 2000; Parida et al. 2007, 2008).
Klein and Itai (1989) found that stress relief did not result
in a rapid destruction of proline, and that leaf proline levels
correlated well with stomatal resistance, suggesting that
proline itself may be involved in the recovery of stomata
and the photosynthetic apparatus by way of stored water
(Dichio et al. 2006). Subsequently, free proline increases
stopped immediately upon rehydration, and thereafter,
levels of proline declined (Stewart 1972). However, Souza
et al. (2004) reported that increases in proline level were
small, and their onset was delayed after stress imposition,
so that it may rather be a consequence and not a stress-
induced beneficial response.
In conclusion, Leaf RWC, Pn, gs, and WUE decreased,
while membrane permeability, lipid peroxidation, Ci, sol-
uble protein, and free proline increased during three soil
drought periods for both psammophytes. These physio-
logical characteristics were recovered to the control levels
following rewatering for 4 days. However, activities of
SOD, CAT, and POD were induced continuously under soil
drought conditions, and remained higher than those in the
control throughout the whole experiment, which agrees
with our hypothesis that drought hardening activates
defensive systems of both psammophytes continuously.
Decreasing level of leaf RWC and increasing levels of leaf
membrane permeability and lipid peroxidation were sup-
pressed with increasing the number of drought cycles,
suggesting that drought hardening alleviate damages of
both psammophytes and improve their drought tolerance
and acclimation to soil drought conditions in the future.
Additionally, the photosynthesis decreased more slowly in
the subsequent drought cycles than in the first cycle,
allowing both psammophytes to maximize assimilation in
response to repeated soil drought conditions. Thus, both
psammophytes acclimatize themselves to repeated soil
drought.
It is regrettable that only data investigated was procured
during each soil rewatering trial, making the rewatering
data insufficient. In view of this fact, the recovery pro-
cesses of the two psammophytes under investigation during
the rewatering treatment remain unanswered. As a result,
recovery patterns require further investigation in order to
clarify the capacity to withstand and survive extreme soil
drought conditions.
Acknowledgments Authors thank all the members of Naiman
Desertification Research Station, Chinese Academy of Sciences
(CAS). We wish to thank anonymous reviewers for valuable com-
ments on the manuscript. This paper was financially supported by the
National Basic Research Program of China (2009CB421303), the
Knowledge innovation Programs of the Chinese Academy of Sciences
(KZCX2-YW-431), the National Nature Science Foundation of China
(40601008), National Key Technologies Support Program of China
(2006BAC01A12, 2006BAD26B02).
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